Intact adult hippocampi and primary hippocampal cultures from wild-type and Girk2 −/− mice were homogenized in RIPA lysis buffer containing Halt phosphatase and protease inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 4 °C for 20 min at 16,000 × g. The supernatant was taken and protein concentrations were determined by BCA assay. Equal amounts of protein from each sample were mixed with SDS sample buffer and heated at 85 °C for 10 min. Samples were then separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk/PBS, incubated overnight at 4 °C with rabbit polyclonal antibodies directed against GIRK1 (1:200, Alomone Labs; Jerusalem, Israel), GIRK2 (1:200, Alomone Labs), GIRK3 (1 μg/mL, Frontier Institute Co.; Ishikari, Japan), or β-actin (0.27 μg/mL, Abcam; Cambridge, MA), and diluted in 5% milk/PBS/0.1% Tween 20. The custom GIRK2c antibody was generated and affinity-purified using a peptide corresponding to the unique 11-amino acid C-terminal domain (DVANLENESKV, Pacific Immunology Corp.; Ramona, CA). Membranes were washed with PBS/0.1% Tween 20 and incubated with donkey anti-rabbit (0.2 μg/mL, LI-COR Biotechnology; Lincoln, NE) or donkey
